Recombinase-Based System for Expression of Foreign Proteins Using Adenovirus Vectors

ABSTRACT

In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox sites positioned such that site-specific recombination between lox sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of copending parent application Ser.No. 09/909,414, filed Jul. 17, 2001, which is continuation of parentapplication Ser. No. 09/263,650, filed on Mar. 5, 1999 (U.S. Pat. No.6,379,943).

FIELD OF THE INVENTION

The present invention relates to methods for efficient and reliableconstruction of adenovirus vectors that contain and express foreign DNAand are useful for gene transfer into mammalian cells, for vaccines andfor gene therapy. The vector system described herein is an improvementand modification of the pBHG system, described in U.S. Pat. No.6,140,087 (Graham et al.), a foreign equivalent of which published asWO95/00655, both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

As taught in U.S. Pat. No. 6,140,087, adenoviruses (Ads) can be used asmammalian cell expression vectors, with excellent potential as liverecombinant viral vaccines, as transducing vectors for gene therapy, forresearch, and for production of proteins in mammalian cells.

In the human Ad genome, early region 1 (E1), E3, and a site upstream ofE4 have been utilized as sites for introducing foreign DNA sequences togenerate adenovirus recombinants. In the absence of compensatingdeletions in E1 or E3, a maximum of about 2 kb can be inserted into theAd genome to generate viable virus progeny. The E1 region is notrequired for viral replication in complementing 293 cells, or othercells known to complement E1, and up to 3.2 kb can be deleted in thisregion to generate conditional helper independent vectors with acapacity of 5.0-5.2 kb. In the E3 region, which is not required forviral replication in cultured cells, deletions of various sizes havebeen utilized to generate nonconditional helper independent vectors witha capacity of up to 4.5-4.7 kb. The combination of deletions in E1 andE3 permits the construction and propagation of adenovirus vectors with acapacity for insertions of up to approximately 8 kb of foreign DNA.

The construction of Adenovirus vectors can be performed in many ways.One approach is to cotransfect permissive cells, usually 293 cells, witha shuttle plasmid containing a portion of the left end of the Ad genomeand, most commonly, having the E1 sequences replaced by a foreign DNA,and with DNA isolated from virions cleaved near the left end by asuitable restriction enzyme. Homologous recombination betweenoverlapping viral DNA sequences of the shuttle plasmid and the virionDNA results in production of recombinant viruses containing the foreignDNA. A disadvantage of this method is the need to prepare purified viralDNA. In addition, such methods typically result in the presence ofcontaminating parental virus in the resulting vector preparations, suchas when 100% of the viral DNA is not cleaved, or when the two viral DNAfragments produced by restriction cleavage are rejoined.

Another method has recently been described (Hardy S, Kitamura M,Harris-Stansil T, Dai Y, Phipps M L, “Construction of adenovirus vectorsthrough Cre-lox recombination.” J Virol 1997 March; 71(3):1842-1849; seealso PCT publication WO97/32481 relating to use of site-specificrecombination of virus and helper dependent vectors) which involvesinfection of 293Cre cells (293 cells engineered to express Crerecombinase) with an Adenovirus containing a floxed packaging signal (1)and transfection with a shuttle plasmid containing an ITR, a packagingsignal and an expression cassette followed by a lox site, orcotransfection of 293Cre cells with purified deproteinized AdenoviralDNA and a shuttle plasmid. According to that method, Cre-mediatedexcision of the packaging signal from virus followed by site-specificrecombination with the lox site in the shuttle plasmid produces arecombinant vector containing the expression cassette. However, as Creaction is not 100% efficient, the resulting virus preparations remaincontaminated with parental virus, and must be passaged in 293Cre cellsto eliminate the contaminating parental virus. A further disadvantage ofthis method is that it requires use of an infectious virus or DNAextracted from a virus as one of the starting materials, and is thusless attractive for commercial distribution than kits containing onlybacterial plasmid DNA. Furthermore, the parental virus can recombinewith Ad E1 sequences present in 293 cells, resulting in a viruscontaining a wild-type packaging signal and a wild-type E1 region. Suchrecombinant virus has the propensity to overgrow the original vector,leading to contamination of subsequent vector preparations withnon-attenuated E1 expressing Ads.

One of the most frequently used and most popular methods forconstruction of adenovirus vectors is based on “the two plasmid method”(see Bett et al., “Packaging capacity and stability of human adenovirustype 5 vectors,” J. Virol. 67:5911-5921, 1993), whereby suitable hostcells (typically 293 cells) are cotransfected with two plasmids thatseparately are incapable of generating infectious virus, but which, whenrecombined within the transfected cell by homologous recombination, cangenerate replicating virus. The most widely used plasmids of this typeare described in U.S. Pat. No. 6,140,087, hereby incorporated byreference. That system has advantages over other methods using virusesor viral DNA as components since only easily-prepared plasmid DNAs areneeded, and there is no background of parental virus that couldcontaminate the final vector isolates. Furthermore, the plasmids are notonly easy and inexpensive to produce by those skilled in the art, butcan be easily stored and transported, making them convenient forcommercial distribution, (i.e. particularly when precipitated withethanol or when lyophilized, these vectors do not require a cold chainfor distribution). However, although this currently available system hasproven utility and is widely used, the efficiency of virus production byhomologous recombination can be low and variable, and the system cannotalways be used easily by those not skilled in the art.

As demonstrated in (Anton, M. and Graham, F. L. “Site-specificrecombination mediated by an adenovirus vector expressing the Crerecombinase protein: a molecular switch for control of gene expression,”J. Virol. 69:4600-4606, 1995), and as described also in parentapplication Ser. No. 08/486,549 (“Adenoviruses for control of geneexpression”, hereby incorporated by reference), provision of Crerecombinase in Ad-infected cells can catalyse excision or rearrangementof viral DNA sequences that contain the target sites (lox P) forCre-mediated site-specific recombination. Such techniques are applied innew ways in the present invention disclosure to provide a long-neededadvancement in the art of adenoviral vector production.

SUMMARY OF THE INVENTION

In the present invention, viruses, plasmids or both are constructedwhich contain viral DNA and lox P sites positioned such thatsite-specific recombination between lox P sites in separate plasmidsresults in generation of infectious viral DNA at high-efficiency incotransfected host cells that have been engineered to express the Crerecombinase. Such cells (293Cre cells) have been described by Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. and Graham, F. L.“A new helper-dependent adenovirus vector system: removal of helpervirus by Cre-mediated excision of the viral packaging signal,” Proc.Natl. Acad. Sci. U.S. 93: 13565-13570, 1996, by Chen, L., Anton, M. andGraham, F. L., “Production and characterization of human 293 cell linesexpressing the site-specific recombinase Cre,” Somat. Cell and Molec.Genet. 22: 477-488, 1996, in U.S. patent application Ser. No.08/473,168, and in PCT publication WO96/40955, hereby incorporated byreference for this purpose. Because of the high-efficiency andspecificity of the Cre enzyme, suitably engineered plasmids can bereadily recombined to produce infectious virus at high-efficiency incotransfected 293 cells, without, at the same time, producing acontaminating parental adenovirus, with the attendant problems forremoval thereof.

In one embodiment, this invention provides a two plasmid system whereinhomologous recombination via cellular enzymes is replaced by sitespecific recombination, via a recombinase such as Cre, to join (withhigh efficiency) two DNAs that separately are noninfectious to form aninfectious DNA molecule. One application of the techniques disclosedherein is the isolation of “first generation” vectors with insertions offoreign DNA in E1. Such applications utilize a series of plasmids suchas pBHG10lox (see FIG. 1, and variations and equivalents thereof), andvarious shuttle plasmids containing the left ITR, a packaging signal, anexpression cassette, and a lox or other recombinase recognition site.Another application is in a sense the mirror image. Using a plasmid suchas pFG173lox, sequences are rescued into the right end of the viral DNA,into E3 or into sites rightward of E3. The most important applicationsof this technology will likely be rescue of mutations into the fibregene located immediately rightward of E3 (FIG. 9) (fibre is importantbecause it is a major ligand for binding to cellular receptors) but onecan also rescue mutations, deletions, insertions and other modificationsin E4 genes (located between fibre and the right ITR) or use the methodto rescue inserts of foreign DNA into E3 (cotransfection of a plasmidsuch as that depicted in FIG. 11 with pFG173lox). Note that the plasmidpFG173lox has a deletion of fibre, but E4 sequences could just as wellbe deleted as well as or instead of fibre. Note also that lox sitescould be inserted at other locations in the Ad genome to enable therescue of mutations engineered in other viral genes besides those offibre or E4, or rescue of DNA inserts into other sites.

In a further embodiment of this invention, DNA-TP complexes are utilizedto combine the high efficiency of Cre-lox recombination with the highinfectivity of DNA-TP. While the rescue of infectious virus via Cremediated recombination is surprisingly efficient compared to homologousrecombination, and is more than adequate to produce viral vectors and tointroduce mutations into the viral genome for most applications, theremay be certain applications for which even higher efficiencies aredesirable or necessary. It is known by those skilled in the art that theinfectivity of adenovirus DNA is up to 100 fold higher if the virion DNAis extracted and purified by methods that leave intact the terminalprotein (TP) that is normally linked to the 5′ end of each strand of theduplex Ad DNA molecule (Sharp P A, Moore C, Haverty J L, “Theinfectivity of adenovirus 5 DNA-protein complex,” Virology 1976December; 75(2):442-456, Chinnadurai G, Chinnadurai S, Green M,“Enhanced infectivity of adenovirus type 2 DNA and a DNA-proteincomplex.” J Virol 1978 April; 26(1):195-199). For rescue of cassettes,the two plasmid system is more than sufficiently efficient, especiallywith the approximately 30-fold enhancement demonstrated herein forCre-lox mediated recombination over homologous recombination, andconsequently would be preferred for most purposes. However, there may betimes when even higher efficiencies are required, as when, for example,one wishes to develop a library of fibre mutations (a large number ofdifferent viruses—the more the better). Then the chore of preparingDNA-TP might be worthwhile and could be accomplished by those skilled inthe art. Thus, an aspect of the present invention includes thecombination of the Cre-lox recombination with the high specificinfectivity of adenoviral DNA-TP complexes.

Therefore, it is an object of the present invention to provide a highlyefficient, reliable, and simple method for isolation of viral vectorsbased on site-specific recombination catalysed by a site-specificrecombinase, such as but not limited to the Cre recombinase, rather thanrelying on homologous recombination, which depends on normal cellularrecombinase pathways.

It is a further object of this invention to use Cre-lox-mediatedrecombination and known two plasmid vector production systems to providea simple method for introducing mutations or other modifications ofviral genes into any desired location in the viral genome.

It is a further object of this invention to provide a simple and usefulsystem by which adenovirus cloning vectors may be developed.

It is a further object of this invention to provide a kit for productionof adenoviral vectors for vaccine and gene-therapeutic applicationswhich relies on site-specific recombination, rather than homologousrecombination, and which does not require a cold-chain for distribution.

A further object of this invention is to provide a system whereby thehigh-efficiency of Cre-lox recombination is combined with enhancedinfectivity achieved when adenovirus-TP complexes are utilized.

Further objects of this invention will become apparent from a review ofthe complete disclosure and the claims appended hereto.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagrammatic representation showing a method for isolationof an Ad vector containing an expression cassette in E1 using theCre/lox recombination system. pBHG10lox comprises a circularized form ofthe Ad genome with a deletion of the E1 region including the packagingsignal and a bacterial plasmid origin of replication and an ampicillinresistance gene. The plasmid has a loxP site near the 5′ end of the pIXgene of the Ad genome and a deletion of E3 sequences. The “shuttleplasmid” contains an ITR of the virus genome and a packaging signal, apolycloning site for insertion of a foreign DNA (eg bacterialβ-galactosidase (LacZ)) and a loxP site inserted in the same relativeorientation as the loxP site in pBHG10lox. Cotransfection of these twoplasmids into 293Cre cells that express Cre results in Cre-mediatedrecombination and formation of joint molecules that generate infectiousviruses containing the foreign DNA insert.

FIG. 2 illustrates a cotransfection experiment similar to that depictedin FIG. 1 except that the shuttle plasmid contains Ad sequences 3′ ofthe lox site that overlap (are homologous) with viral sequences inpBHG10lox to the right of the lox site. Therefore an Ad vectorcontaining an expression cassette in E1 can be generated by Cre/loxrecombination when the two plasmids are cotransfected into 293Cre cellsor alternatively by homologous recombination between overlappingsequences. The shuttle plasmid in the illustration permits a comparisonof the efficiency obtainable from the two recombination modes.

FIG. 3 illustrates four sets (pairs) of oligonucleotides used in variouscloning procedures. The oligonucleotides are annealed prior to use toproduce the double stranded DNA segments illustrated. Three of theoligonucleotide pairs contain loxP, the recognition site for Crerecombinase as well as one or more restriction endonuclease sites usedfor diagnostic purposes or for subsequent cloning steps. One of theoligonucleotide pairs contains several restriction endonuclease sitesand was used to introduce a polycloning site into various shuttleplasmids.

FIG. 4A illustrates the construction of a plasmid, derived from pBHG10(Bett, A. J., Haddara, W., Prevec, L. and Graham, F. L “An efficient andflexible system for construction of adenovirus vectors with insertionsor deletions in early regions 1 and 3.” Proc. Natl. Acad. Sci. US 91:8802-8806, 1994., available from Microbix Biosystems), wherein a loxPsite is inserted at the 3′ end of an E1 deletion and 5′ (upstream) ofthe pIX gene. PBHG10lox was constructed by replacing the 4604 bpBst1107I fragment from pBHG10 with the 2326 bp EcoRV/Bst1107I fragmentfrom pΔE1sp1Alox (see FIG. 5). Foreign DNA sequences can be insertedinto the unique PacI site of pBHG10lox for rescue of genes in E3.

FIGS. 4B-1 and 4B-2 illustrate the construction of a plasmid,pBHGdX1Plox, containing a modified E3 deletion (taken from pFG23dX1) anda lox site 5′ of the pIX gene. The plasmid pFG23dX1P was constructed byinserting an oligonucleotide containing a PacI site (SEQ ID NO:9;AB14566; 5′-CTAGCTTAATTAAG-3′; this oligo self anneals to produce adouble stranded DNA with 5′ overhangs that hybridize to overhangsgenerated by XbaI cleavage) into the XbaI site of pFG23dX1. Theresulting plasmid, pFG23dX1P, is identical to pFG23dX1 except that theunique XbaI site at nt 11392 is changed to a unique Pac I site. Theplasmid pNG17 was constructed by cloning the 6724 bp SpeI/ClaI fragmentfrom pBHG10lox into pBluescript. The plasmid pNG17dX1P was constructedby replacing the 1354 bp SpeI/NdeI fragment from pNG17 with the 2143 bpSpeI/NdeI fragment from pFG23dX1P. Finally, the plasmid pBHGdX1Plox wasconstructed by replacing the 6724 bp SpeI/ClaI fragment from pBHG10loxwith the 7513 bp SpeI/ClaI fragment from pNG17dX1P. pBHGdX1Plox thuscontains a modified E3 region such that the deletion of E3 sequences isthat of the parental plasmid pFG23dX1 (a deletion of 1878 bp) ratherthan the larger deletion of the other parental plasmid pBHG10lox.

FIG. 4C illustrates the construction of pBHGE3lox, a plasmid derivedfrom pBHGE3 and pBHG10lox constructed by replacing the 6724 bp SpeI/ClaIfragment from pBHG10lox with the 9377 bp SpeI/ClaI fragment from pBHGE3.PBHGE3lox contains a complete E3 region for isolation of viral vectorsthat retain a wild-type E3.

FIG. 5 illustrates the construction of shuttle plasmids derived frompΔE1SP1A and pΔE1SP1B wherein a loxP site is introduced 3′ of thepackaging signal. The plasmids pΔE1sp1Alox and pΔE1sp1Blox wereconstructed by inserting an oligonucleotide bearing a loxP site(comprised of SEQ ID NO:1 and SEQ ID NO:2 annealed oligonucleotidesequences, also identified as AB3233 and AB 3234) into the BglII site ofp E1sp1A. Subsequent digestion with Nru I and partial Sca I digestionfollowed by ligation generated pΔE1SP1AloxΔ and pΔE1SP1BloxΔ.

FIG. 6A illustrates the construction of pMH4lox, pMH4loxΔ andpMH4loxΔlink plasmids that contain lox sites and a promoter andpolyadenylation signal and polycloning sites for insertion of foreignDNA to produce expression cassettes in which transcription is driven bythe murine cytomegalovirus immediate early gene promoter. Plasmid pVDB3(see FIG. 6 a) is derived from pMH4 but contains a pUC based origin ofreplication rather than a pBR322 origin. It contains Ad5 sequences fromm.u. 0-15.8 with E1 sequences deleted between m.u. 1 and 9.8 andsubstituted with an expression cassette: a 0.5 kbp (−491 to +36)fragment of the MCMV IE promoter, unique restriction enzyme sites forcloning (Eco RI, Nhe I, Bam HI and Sal I) followed by an SV40polyadenylation signal. To make pMH4lox, a loxP linker (SEQ ID NO:1 andSEQ ID NO: 2; AB3233/3234) was introduced into the BglII site of pVDB3.Ad5 sequences m.u. 9.8-15.8 were deleted from pMH4lox by digesting withHind III, treating with the Klenow fragment of E. coli DNA polymerasethen partially digesting with Sca I followed by self-ligation. Theresulting shuttle plasmid, pMH4loxΔ, can be used with pBHG10lox toproduce Ad vectors via Cre/lox mediated recombination. To make pMH4loxΔa more flexible plasmid for cloning purposes, a linker (SEQ ID NO:3 andSEQ ID NO:4; AB14626/14627) containing a different multiple cloningregion was introduced between the Eco RI and Sal I sites resulting inpMH4loxΔlink.

FIG. 6B illustrates the construction of plasmid pVDB3 derived from pMH4but containing a pUC based origin of replication rather than a pBR322origin. A PvuI to Bst 11071 fragment from pMH4 (Microbix Biosystems) wasligated to a Bst 11071 to Pvu I fragment from pD47E1 containing a pUCbased (pNEB193, New England Biolabs) origin of plasmid DNA replicationto generate pVDB3.

FIG. 7 illustrates construction of HCMV loxP plasmids, pCA13loxΔ andpCA14loxΔ, in which transcription of foreign genes is regulated by thehuman cytomegalovirus immediate early gene promoter. The plasmidspCA13(ΔBglII) and pCA14(ΔBglII) were generated by digesting pCA13 andpCA14 partially with BglII, Klenowing and self-ligating. A syntheticloxP oligonucleotide (SEQ ID NO:1 and SEQ ID NO:2; AB3233/3234) wasintroduced into the unique BglII sites of pCA13(ΔBglII) andpCA14(ΔBglII) producing pCA13lox and pCA14lox respectively. Ad5sequences, m.u. 9.8-15.8, were removed from pCA13lox and pCA14lox bycutting each plasmid with NruI and partially digesting each with ScaIfollowed by self ligation.

FIG. 8A is a diagrammatic representation of a method for constructingpCA36loxΔ a shuttle plasmid containing the leftmost approximately 340 ntof Ad5, an expression cassette encoding β-galactosidase, and a lox Psite for rescue of the LacZ gene into adenovirus vectors. A syntheticloxP site (SEQ ID NO:1 and SEQ ID NO: 2; AB3233/3234) was introducedinto the Bgl II site of pCA36 resulting in pCA36lox. This plasmid wasthen digested with Nru I and partially digested with Sca I, a 7646 bpfragment was gel purified and self ligated yielding pCA36loxΔ.

FIG. 8B is a diagrammatic representation of a means to isolateadenoviral vectors containing an expression cassette by cotransfectionof 293Cre cells with (a) AdLC8c DNA-TP complex having covalently boundterminal protein (TP) linked to the 5′ ends of Adenoviral DNA and (b) ashuttle plasmid containing an expression cassette and a lox P site.Cre-mediated excision of the floxed packaging signal of AdLC8c rendersthe AdLC8c genome defective for packaging. A second Cre-mediatedrecombination event between the lox sites in the shuttle plasmid and theAdLC8c genome results in a vector with a packaging signal, the foreignDNA insert, and a single lox site.

FIG. 8C is a diagrammatic representation of a means to isolateadenoviral vectors containing an expression cassette by cotransfectionof 293Cre cells with restricted AdLC8c DNA-TP and a shuttle plasmidcontaining an expression cassette and a lox P site. AdLC8c DNA-TP iscleaved with an endonuclease such as Asu II or Swa I that recognizeunique restriction enzyme sites between the lox sites flanking 2.Cleavage of viral DNA with restriction enzymes prior to cotransfectionreduces the infectivity of parental virus DNA and when combined with thehigh-efficiency of Cre-mediated recombination results in high-efficiencyof vector isolation in cotransfected 293Cre cells as illustrated.Rejoining of parental DNA fragments and generation of infectiousparental virus rather than the desired vector is avoided because of theaction of Cre on the floxed packaging signal in AdLC8c. However, whenthe viral DNA-TP complex is cut with a restriction enzyme asillustrated, the level of Cre-mediated recombination is sufficientlyhigh that most, if not all, progeny viruses result from recombinationbetween the shuttle plasmid and the large DNA-TP fragment. Therefore,the left-most lox site of AdLC8c and equivalent vectors is notessential.

FIG. 8D is a diagrammatic representation of a method for constructingshuttle plasmids expressing Cre. The Cre expression cassette wasobtained from the plasmid pLC2 (Chen, L., Anton, M. and Graham, F. L.,“Production and characterization of human 293 cell lines expressing thesite-specific recombinase Cre,” Somat. Cell and Molec. Genet.22:477-488, 1996), as a 2175 bp BglII fragment which was end-modifiedwith Klenow DNA polymerase and inserted into the EheI site of pCA36loxΔto generate pCA36loxΔCreR and pCA36loxΔCreT. The plasmid pCA36loxΔCreITRwas constructed by replacing the 1402 bp ScaI/KpnI fragment inpCA36loxΔCreT with the 2753 bp ScaI/KpnI fragment from the plasmidpRP1029. Plasmid pCA36loxΔCreITR contains ITR junctions which are knownto be functionally capable of generating replicating linear Ad DNAmolecules (Graham, F. L., “Covalently closed circles of human adenovirusDNA are infections,” The EMBO J. 3, 2917-2922, 1984).

FIG. 8E provides a schematic representation of a cotransfectionexperiment wherein a pBHG10lox plasmid and a “Lox” shuttle plasmidexpressing Cre are introduced into 293 cells in order to generate Adexpression vectors, without having to use cells which stably expressCre. This technique is applicable to any cell type suitable for Advector generation, including but not limited to 293 cells, and PER-C6cells (Fallaux et al., Hum. Gene Ther. 1998, Sep. 1; 9(13):1909-17), 911cells (Fallaux et al., Hum. Gene Ther. 1996 Jan. 20; 7(2):215-222), orother cells. A shuttle plasmid such as pCA36loxΔCreITRof FIG. 8 c isalso suitable for generation of an adenovirus vector.

FIG. 8F. Demonstrates the construction of an Ad genomic plasmid encodingCre. The plasmid pBHG10loxΔ was constructed by collapsing pBHG10lox withSpeI and PshAI. The Cre expression cassette, taken from the plasmid pLC2as a 2175 bp BglII fragment, was inserted into the BamHI site ofpBHG10loxΔ to generate pBHG10loxΔCreR and pBHG10loxΔCreL. The 1238 bpBst1107I/PacI fragment from pBHG10loxΔCreR and pBHG10loxΔCreL wasreplaced with the 22380 bp Bst1107I/PacI fragment from pBHG10lox togenerate pBHG10loxCreR and pBHG10loxCreL, respectively.

FIG. 9A is a diagrammatic representation of a method for rescuing fibremutations into infectious virus using Cre-lox recombination. PlasmidpFG173lox is derived from pFG173 which is a bacterial plasmid containingmost of the Ad5 genome but from which sequences have been deleted(represented by “deletion” in the diagram) that render the plasmidnoninfectious. The sequences are substituted with bacterial DNAcontaining an antibiotic resistance gene and a bacterial plasmid originof DNA replication. A lox site upstream (leftward in the conventionalmap of the Ad genome) of the deletion/substitution is inserted in theplasmid for Cre-mediated recombination with a similar lox site in ashuttle plasmid containing the right region of the viral genome fromapproximately 85 mu to approximately 100 mu and including most or all ofthe right ITR. Recombination as illustrated generates an infectiousvirus containing sequences representing the left approximately 78 mu ofthe Ad genome derived from pFG173lox and sequences from approximately85-100 mu derived from the shuttle plasmid.

FIG. 9B is a diagrammatic representation of a method for constructing aplasmid containing a lox site and ampicillin resistance genesubstituting for the fibre gene. Starting with pAB14lox whoseconstruction is described in FIG. 14, the DNA sequences between the ClaI site and the Blp I site containing fibre are substituted with a DNAsegment containing the ampicillin resistance gene and a plasmid originof DNA replication. The NdeI to Ssp I DNA fragment from pCA14 (MicrobixBiosystems) containing ampicillin resistance gene and plasmid origin ofDNA replication is treated with Klenow DNA polymerase and ligated with asimilarly treated Blp I to ClaI fragment of pAB14lox to generate theampicillin and kanamycin doubly resistant, fibre gene deleted,pAB14loxΔ.

FIG. 9C is a diagrammatic representation of a method for combining theplasmid of FIG. 9 a with pFG173 to produce pFG173lox for rescuing fibreor E4 mutations into infectious virus using Cre-lox recombination. Theplasmid pAB14loxΔ is treated with restriction enzymes that cut in andaround the kanamycin resistance gene and pFG173 is similarly digestedwith Eco RI as illustrated. Transformation of E. coli with thefragmented DNA from the two plasmids results in formation of areplicating plasmid in which the sequences in and around the shadedportion indicated in pFG173 are substituted with corresponding sequencesfrom pAB14loxΔ by homologous recombination (Chartier C, Degryse E,Gantzer M, Dieterle A, Pavirani A, Mehtali M. Efficient generation ofrecombinant adenovirus vectors by homologous recombination inEscherichia coli. J Virol 1996 July; 70(7):4805-4810).

FIG. 10 is a diagrammatic representation of method for constructing aplasmid containing the right approximately 40% of the virus genomewherein a lox P site has been inserted near the 5′ end of the fibregene. The plasmid pFG23dX1 contains the right 40% of the Ad5 genomecloned into the bacterial plasmid pBR322, and has a deletion of an XbaIfragment from nt 28,589 (79.6 mu) of the wt Ad5 sequence to nt 30470 (mu84.8) leaving a unique XbaI site suitable for insertion of a loxP site.A loxP site comprised of two synthetic oligonucleotides (SEQ ID NO:5 andSEQ ID NO:6; AB6920/AB6921, FIG. 3) was ligated into the Xba I site ofpFG23dX1 to generate pFG23dX1lox which contains a loxP site upstream ofthe sequences encoding fibre. Finally, pFG23dX1lox was further modifiedby deletion of viral sequences between a unique Bst11071 site and aBsiW1 site immediately 5′ of the lox P site to generate pFG23dX1loxc.

FIG. 11 illustrates a pFG23dX1lox plasmid with an expression cassetteencoding bacterial β-galactosidase inserted into the Cla I site betweenthe lox P site and the fibre gene.

FIG. 12 is a diagrammatic representation showing rescue of a fibremutation into a virus genome by cotransfection of 293Cre cells withDNA-TP of an Adfloxed fibre and a plasmid containing a lox P site 5′ ofa (optionally mutated) fibre gene. Viral DNA-TP complex extracted fromvirus preparations of Adfloxfibre (FIG. 15) and plasmid DNA (pFGdX1lox)optionally carrying a mutated fibre gene are cotransfected into 293Crecells to produce a recombinant virus expressing the optionally mutatedfibre. If desired, viral DNA can be prepared so that the terminalprotein remains linked to the ends of the virion DNA as indicated.

FIG. 13 is a diagrammatic representation showing rescue of a foreign DNAsequence into a virus genome by cotransfection of 293Cre cells withDNA-TP of an Adfloxed fibre and a plasmid containing a lox P site, and aforeign DNA inserted 5′ of the fibre gene. Cotransfection of cells withAdfloxfibre DNA-TP and pFG23dX1LacZlox results in production of a vectorcarrying the foreign (e.g. lacZ) gene inserted upstream of fibre. Asnoted above in the description of FIG. 8B, the right-most lox sitedepicted in the Adfloxed fibre genome can be omitted if the DNA-TP isdigested with one or more restriction enzymes which cut rightward of thelox site located 5′ of fibre.

FIG. 14 is a diagrammatic representation showing construction of aplasmid containing a fibre gene with flanking lox P sites. Plasmid pAB14(described in: Bett, A. J., Prevec, L., and Graham, F. L. Packagingcapacity and stability of human adenovirus type 5 vectors. J. Virol. 67:5911-5921, 1993.) contains Ad sequences from approximately mu 0 to 1.0,10.6 to 16.1, 69.0 to 78.3, and 85.8 to 100. The plasmid has unique XbaIand BlpI restriction sites suitable for insertion of syntheticoligonucleotides containing lox P sites as illustrated. PAB14flox wasconstructed by first inserting a lox site into the XbaI site that isupstream of fibre to produce pAB14lox. Subsequently a second lox sitewas inserted into the unique Blp I site in pAB14 which is locatedbetween the 3′ terminus of the fibre gene and the coding regions of E4genes (pAB14flox: fibre flanked by lox sites).

FIG. 15 is a diagrammatic representation showing isolation of a virusgenome containing lox P sites flanking the fibre gene (floxed fibre).Cotransfection of pAB14flox with pFG173 (described in Hanke, T., Graham,F. L., V. Lulitanond and D. C. Johnson. Herpes simplex virus IgG Fcreceptors induced using recombinant adenovirus vectors expressingglycoproteins E and I. Virology 177: 437-444, 1990. PFG173 is availablefrom Microbix Biosystems) generates a virus containing a floxed fibregene, Adfloxfibre.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One embodiment of the present invention provides a bacterial plasmidcomprising an antibiotic resistance gene and origin of replication forreplication of said plasmid in host cells and further comprising acircularized modified human adenovirus genome that contains sequencesthat can be recognized and acted upon by the site-specific recombinasesuch as Cre, FLP or like recombinases. Said bacterial plasmid isdesigned to be unable to generate infectious adenovirus by virtue of adeletion of viral DNA sequences, such as the packaging signal, which isnormally located at the left end of wild-type Ad DNA, and which isessential for virus replication. Alternatively, formation of infectiousvirus may be prevented by the insertion of DNA (“stuffer DNA”) such thatthe overall size of the resulting virus DNA exceeds the upper packaginglimit for Ad virions (approximately 38 kb). Deletion of the pIXsequences from the Ad sequences makes the size-limitation of thepackaging limitation more stringent, unless complementing cells whichexpress the pIX gene product are used. Optionally, certain additionalviral DNA sequences may be deleted, such as sequences from E3, which canin any event be omitted from the viral genome without preventing a viralgenome from replicating in such cells as may be permissive forreplication of said viral genome in the form of infectious virus.

Another embodiment of the invention provides a second bacterial plasmid,known as a “shuttle” plasmid, comprising minimally approximately 340base pairs from the left end of the Ad5 genome, including the left endterminal repeat (ITR) sequences of said genome and the packaging signalsequences thereof, optionally a promoter, and optionally a foreign DNAencoding a protein and optionally a polyadenylation signal, and a loxsite (various lox sites are known in the art, including, but not limitedto loxP, lox511, lox514, loxPsym, and mention of any one of these sitesincorporates the mention of the other lox sites). The promoter, foreigngene and poly adenylation signal are referred to herein collectively asan “expression cassette”. Co-transfecting 293Cre cells with said shuttleplasmid and the plasmid of the first embodiment of the invention resultsin recombination between said plasmids and rescue of said expressioncassette into an infectious viral vector by action of said Crerecombinase.

It will be appreciated that the term “bacterial plasmid” is not meant tobe limiting, since one skilled in the art would recognize that othertypes of DNA could be recombined by the Cre recombinase with equalefficiency. For example, the Cre recombinase could be expressed in yeastcells to allow for high-efficiency recombination between yeastartificial chromosomes (YAC's) harboring an Ad genome, or, similarly, inbacteria, to allow for Cre-mediated recombination between cosmids orbacteriophage genomes harboring Ad sequences. Similarly, expression ofCre in mammalian cells could be used to allow for efficientrecombination between two or more infectious Ad vectors, between an Advector and a bacterial plasmid, between an adenoviral genome and alinear DNA fragment and the like.

A third embodiment of the invention provides a mammalian cell line, suchas a human cell line, that provides the Cre recombinase enzyme.Alternatively, Cre may be provided by an Ad5 derived vector thatexpresses the Cre protein in suitable cells or Cre may be provided by athird plasmid encoding Cre or optionally Cre could be expressed from anexpression cassette inserted into one of the two plasmids used in thetwo plasmid rescue system. Alternatively, Cre could be expressed inother species, for example bacteria or yeast, to allow for recombinationand generation of recombinant Ad genomes in said species. Alternatively,Cre could be provided as a pure or crude protein extract from expressionin a variety of species for recombination of said bacterial plasmids invitro. One skilled in the art would recognize that other recombinasesystems are available which could catalyse similar recombination eventsin place of Cre, for example, not meant to be limiting, the yeast FLPrecombinase recognizes and recombines FRT target sites and is thereforeexpected to provide functions similar to those described herein withreference to Cre and its loxP target sites.

A fourth embodiment of the invention provides an adenovirus or a plasmidcontaining adenovirus DNA wherein a segment of the viral DNA such as,but not limited to, the region encoding fibre is flanked by lox P sites.

A fifth embodiment of the invention provides an adenovirus or a plasmidcontaining adenovirus DNA wherein a segment of the viral DNA such as,but not limited to, the region encoding fibre is deleted and substitutedby a lox P site.

A sixth embodiment of the invention provides a plasmid containing aportion of the viral genome including a segment of viral DNA comprising,for example, fibre coding sequences wherein a single lox P site isembedded upstream of fibre coding sequences such that Cre-mediatedrecombination between said plasmid DNA and the plasmid of the fifthembodiment results in production of an infectious viral genome.Optionally the fibre gene in said plasmid may be modified by mutation,insertion or deletion of portions of the fibre coding sequences. Similarplasmids can be constructed that have lox P sites at other locations,depending on the viral DNA segment that is to be manipulated bysite-specific recombination. For example, a site exists in the Ad genomebetween the coding sequences of fibre and the coding sequences of E4that is suitable for insertion of DNA.

In a seventh embodiment of the invention, plasmids containing adenovirussequences and lox sites are recombined in the presence of Crerecombinase to generate novel adenovirus mutants containingmodifications of the fibre gene or modifications of other viral genes.

In a preferred embodiment of the present invention, a system isdescribed for the construction of novel Ad vectors, or alteration ofexisting Ad vectors, by the use of a site-specific recombinase.

In a further embodiment of the invention, an infectious viral DNA-TPcomplex is engineered to take advantage of recombinase-mediatedsite-specific recombination and the enhanced level of infectivityachieved through presence of the terminal protein.

It will be appreciated by those skilled in the art that the presentinvention disclosure provides significant advances over techniques knownin the art for generation of adenoviral vectors. First, the efficiencyby which recombinants are produced is enhanced through use ofsite-specific recombination, rather than relying exclusively onhomologous recombination. Nonetheless, based on the present disclosure,those skilled in the art will appreciate that homologous recombinationmay be used in combination with the site-specific methodology describedherein. This invention further advances the art in that it facilitatesuse of vectors which are themselves non-infectious and stable. Further,by use of the methods disclosed herein, rapid production of recombinantvirus is facilitated wherein every virus produced is a recombinantvirus, as opposed to known methods wherein a starting virus is used in asite-specific recombination wherein substantial levels ofnon-recombinant starting virus remain in the preparation which has tothen be serially passaged to remove the contaminating starter virus. Asa result of this enhanced efficiency, while it may in many instances bedesirable to colony or plaque-purify the results of a givencotransfection, because all viruses produced according to thisembodiment of the instant technique are recombinants, plaquepurification is not absolutely required. Accordingly, the instant methodprovides the option of rapid production of recombinants and screening ofproducts, in a “shot-gun” approach, which will provide significant laborand time savings to those skilled in the art.

In reviewing the detailed disclosure which follows, it should be bornein mind that any publications referenced herein are hereby incorporatedby reference in this application in order to more fully describe thestate of the art to which the present invention pertains.

It is important to an understanding of the present invention to notethat all technical and scientific terms used herein, unless otherwisedefined, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. The techniques employed herein arealso those that are known to one of ordinary skill in the art, unlessstated otherwise.

Reference to particular buffers, media, reagents, cells, cultureconditions and the like, or to some subclass of same, is not intended tobe limiting, but should be read to include all such related materialsthat one of ordinary skill in the art would recognize as being ofinterest or value in the particular context in which that discussion ispresented. For example, it is often possible to substitute one buffersystem or culture medium for another, such that a different but knownway is used to achieve the same goals as those to which the use of asuggested method, material or composition is directed.

The terms used herein are not intended to be limiting of the invention.For example, the term “gene” includes cDNAs, RNA, or otherpolynucleotides that encode gene products. “Foreign gene” denotes a genethat has been obtained from an organism or cell type other than theorganism or cell type in which it is expressed; it also refers to a genefrom the same organism that has been translocated from its normal situsin the genome. In using the terms “nucleic acid”, “RNA”, “DNA”, etc., wedo not mean to limit the chemical structures that can be used inparticular steps. For example, it is well known to those skilled in theart that RNA can generally be substituted for DNA, and as such, the useof the term “DNA” should be read to include this substitution. Inaddition, it is known that a variety of nucleic acid analogues andderivatives is also within the scope of the present invention.“Expression” of a gene or nucleic acid encompasses not only cellulargene expression, but also the transcription and translation of nucleicacid(s) in cloning systems and in any other context. The term“recombinase” encompasses enzymes that induce, mediate or facilitaterecombination, and other nucleic acid modifying enzymes that cause,mediate or facilitate the rearrangement of a nucleic acid sequence, orthe excision or insertion of a first nucleic acid sequence from or intoa second nucleic acid sequence. The “target site” of a recombinase isthe nucleic acid sequence or region that is recognized (e.g.,specifically binds to) and/or acted upon (excised, cut or induced torecombine) by the recombinase. The term “gene product” refers primarilyto proteins and polypeptides encoded by other nucleic acids (e.g.,non-coding and regulatory RNAs such as tRNA, sRNPs). The term“regulation of expression” refers to events or molecules that increaseor decrease the synthesis, degradation, availability or activity of agiven gene product.

The present invention is also not limited to the use of the cell typesand cell lines used herein. Cells from different tissues (breastepithelium, colon, lymphocytes, etc.) or different species (human,mouse, etc.) are also useful in the present invention.

It is important in this invention to detect the generation andexpression of recombinant nucleic acids and their encoded gene products.The detection methods used herein include, for example, cloning andsequencing, ligation of oligonucleotides, use of the polymerase chainreaction and variations thereof (e.g., a PCR that uses 7-deaza GTP), useof single nucleotide primer-guided extension assays, hybridizationtechniques using target-specific oligonucleotides that can be shown topreferentially bind to complementary sequences under given stringencyconditions, and sandwich hybridization methods.

Sequencing may be carried out with commercially available automatedsequencers utilizing labeled primers or terminators, or using sequencinggel-based methods. Sequence analysis is also carried out by methodsbased on ligation of oligonucleotide sequences which anneal immediatelyadjacent to each other on a target DNA or RNA molecule (Wu and Wallace,Genomics 4: 560-569 (1989); Landren et al., Proc. Natl. Acad. Sci. 87:8923-8927 (1990); Barany, F., Proc. Natl. Acad. Sci. 88: 189-193(1991)). Ligase-mediated covalent attachment occurs only when theoligonucleotides are correctly base-paired. The Ligase Chain Reaction(LCR), which utilizes the thermostable Taq ligase for targetamplification, is particularly useful for interrogating late onsetdiabetes mutation loci. The elevated reaction temperatures permits theligation reaction to be conducted with high stringency (Barany, F., PCRMethods and Applications 1: 5-16 (1991)).

The hybridization reactions may be carried out in a filter-based format,in which the target nucleic acids are immobilized on nitrocellulose ornylon membranes and probed with oligonucleotide probes. Any of the knownhybridization formats may be used, including Southern blots, slot blots,“reverse” dot blots, solution hybridization, solid support basedsandwich hybridization, bead-based, silicon chip-based and microtiterwell-based hybridization formats.

The detection oligonucleotide probes range in size between 10-1,000bases. In order to obtain the required target discrimination using thedetection oligonucleotide probes, the hybridization reactions aregenerally run between 20°-60° C., and most preferably between 30°-50° C.As known to those skilled in the art, optimal discrimination betweenperfect and mismatched duplexes is obtained by manipulating thetemperature and/or salt concentrations or inclusion of formamide in thestringency washes.

The cloning and expression vectors described herein are introduced intocells or tissues by any one of a variety of known methods within theart. Such methods are described for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York (1992), which is hereby incorporated by reference. See, also,Ausubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989); Hitt et al, “Construction and propagationof human adenovirus vectors,” in Cell Biology: A Laboratory Handbook,Ed. J. E. Celis., Academic Press. 2^(nd) Edition, Volume 1, pp: 500-512,1998; Hitt et al, “Techniques for human adenovirus vector constructionand characterization,” in Methods in Molecular Genetics, Ed. K. W.Adolph, Academic Press, Orlando, Fla., Volume 7B, pp:12-30, 1995; Hitt,et al., “Construction and propagation of human adenovirus vectors,” inCell Biology: A Laboratory Handbook,” Ed. J. E. Celis. Academic Press.pp:479-490, 1994, also hereby incorporated by reference. The methodsinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors.

The protein products of recombined and unrecombined coding sequences maybe analyzed using immune techniques. For example, a protein, or afragment thereof is injected into a host animal along with an adjuvantso as to generate an immune response. Immunoglobulins which bind therecombinant fragment are harvested as an antiserum, and are optionallyfurther purified by affinity chromatography or other means.Additionally, spleen cells may be harvested from an immunized mouse hostand fused to myeloma cells to produce a bank of antibody-secretinghybridoma cells. The bank of hybridomas is screened for clones thatsecrete immunoglobulins which bind to the variant polypeptides butpoorly or not at all to wild-type polypeptides are selected, either bypre-absorption with wild-type proteins or by screening of hybridoma celllines for specific idiotypes that bind the variant, but not wild-type,polypeptides.

Nucleic acid sequences capable of ultimately expressing the desiredvariant polypeptides are formed from a variety of differentpolynucleotides (genomic or cDNA, RNA, synthetic olignucleotides, etc.)as well as by a variety of different techniques.

The DNA sequences are expressed in hosts after the sequences have beenoperably linked to (i.e., positioned to ensure the functioning of) anexpression control sequence. These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors containselection markers (e.g., markers based on tetracycline resistance orhygromycin resistance) to permit detection and/or selection of thosecells transformed with the desired DNA sequences. Further details can befound in U.S. Pat. No. 4,704,362.

Polynucleotides encoding a variant polypeptide include sequences thatfacilitate transcription (expression sequences) and translation of thecoding sequences such that the encoded polypeptide product is produced.Construction of such polynucleotides is well known in the art. Forexample, such polynucleotides include a promoter, a transcriptiontermination site (polyadenylation site in eukaryotic expression hosts),a ribosome binding site, and, optionally, an enhancer for use ineukaryotic expression hosts, and optionally, sequences necessary forreplication of a vector.

E. Coli is one prokaryotic host useful particularly for cloning DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. Expression vectors are made in these prokaryotichosts which will typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters are used, suchas the lactose promoter system, a tryptophan (Trp) promoter system, abeta-lactamase promoter system, or a promoter system from phage lambda.The promoters typically control expression, optionally with an operatorsequence, and have ribosome binding site sequences, for example, forinitiating and completing transcription and translation.

Other microbes, such as yeast, are used for expression. Saccharomyces isa suitable host, with suitable vectors having expression controlsequences, such a promoters, including 3-phosphoglycerate kinase orother glycolytic enzymes, and an origin of replication, terminationsequences, etc. as desired.

In addition to microorganisms, mammalian tissue cell culture is used toexpress and produce the polypeptides of the present invention.Eukaryotic cells are preferred, because a number of suitable host celllines capable of secreting intact human proteins have been developed inthe art, and include the CHO cell lines, various COS cell lines, HeLacells, myeloma cell lines, Jurkat cells, and so forth. Expressionvectors for these cells include expression control sequences, such as anorigin of replication, a promoter, an enhancer, and necessaryinformation processing sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobin genes, SV40, Adenovirus, Bovine Papilloma Virus, HerpesVirus, and so forth. The vectors containing the DNA segments of interest(e.g., polypeptides encoding a variant polypeptide) are transferred intothe host cell by well-known methods, which vary depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment orelectroporation is useful for other cellular hosts.

The method lends itself readily to the formulation of test kits for usein diagnosis. Such a kit comprises a carrier compartmentalized toreceive in close confinement one or more containers wherein a firstcontainer contains reagents useful in the localization of the labeledprobes, such as enzyme substrates. Still other containers containrestriction enzymes, buffers etc., together with instructions for use.

The methods provided herein for obtention of recombinant Ad vectors aresignificantly different from previously described methods that rely onhomologous recombination catalysed by recombinases in host cells or thatrely on in vitro ligation of viral DNA fragments to produce infectiousviral DNA. For viral DNA replication and packaging of viral DNA intovirion particles, only three regions of the viral DNA are known to berequired in cis. These are the left inverted terminal repeat, or ITR,(bp 1 to approximately 103) the packaging signals (approximately 194 to358 bp) (Hearing and Shenk, 1983, Cell 33: 695-703; Grable and Hearing1992, J. Virol. 64: 2047-2056) and the right ITR. Among the regions ofthe viral genome that encode proteins that function in trans, two havebeen most important in the design and development of adenovirus vectors.These are early region 3 (E3) located between approximately 76 and 86 mu(mu=% distance from the left end of the conventionally oriented genome)and early region 1 (E1) located between approximately 1 and 11 mu. E3sequences have long been known to be nonessential for virus replicationin cultured cells and many viral vectors have deletions of E3 sequencesso that the capacity of the resulting vector backbone for insertion offoreign DNA is thereby increased significantly over that allowable bythe wild-type virus (Bett, A. J., Prevec, L., and Graham, F. L.Packaging capacity and stability of human adenovirus type 5 vectors. J.Virol. 67: 5911-5921, 1993.). E1 encodes essential functions. However,E1 can also be deleted, providing that the resulting virus is propagatedin host cells, such as the 293 cell line, PER-C6 cells, 911 cells, andthe like, which contain and express E1 genes and can complement thedeficiency of E1(−) viruses.

Viruses with foreign DNA inserted in place of E1 sequences, andoptionally also carrying deletions of E3 sequences are conventionallyknown as “first generation” adenovirus vectors. First generation vectorsare of proven utility for many applications. They can be used asresearch tools for high-efficiency transfer and expression of foreigngenes in mammalian cells derived from many tissues and from manyspecies. First generation vectors can be used in development ofrecombinant viral vaccines when the vectors contain and express antigensderived from pathogenic organisms. The vectors can be used for genetherapy, because of their ability to efficiently transfer and expressforeign genes in vivo, and due to their ability to transduce bothreplicating and nonreplicating cells in many different tissues.Adenovirus vectors are widely used in these applications.

There are many known ways to construct adenovirus vectors. As discussedabove, one of the most commonly employed methods is the so called “twoplasmid” technique. In that procedure, two noninfectious bacterialplasmids are constructed with the following properties: each plasmidalone is incapable of generating infectious virus. However, incombination, the plasmids potentially can generate infectious virus,provided the viral sequences contained therein are homologouslyrecombined to constitute a complete infectious virus DNA. According tothat method, typically one plasmid is large (approximately 30,000-35,000nt) and contains most of the viral genome, save for some DNA segment(such as that comprising the packaging signal, or encoding an essentialgene) whose deletion renders the plasmid incapable of producinginfectious virus. The second plasmid is typically smaller (eg5000-10,000 nt), as small size aids in the manipulation of the plasmidDNA by recombinant DNA techniques. Said second plasmid contains viralDNA sequences that partially overlap with sequences present in thelarger plasmid. Together with the viral sequences of the larger plasmid,the sequences of the second plasmid can potentially constitute aninfectious viral DNA. Cotransfection of a host cell with the twoplasmids produces an infectious virus as a result of homologousrecombination between the overlapping viral DNA sequences common to thetwo plasmids. One particular system in general use by those skilled inthe art is based on a series of large plasmids known as pBHG10, pBHG11and pBHGE3 described by Bett, A. J., Haddara, W., Prevec, L. and Graham,F. L: “An efficient and flexible system for construction of adenovirusvectors with insertions or deletions in early regions 1 and 3,” Proc.Natl. Acad. Sci. US 91: 8802-8806, 1994 and in U.S. Pat. No. 6,140,087(hereby incorporated by reference). Those plasmids contain most of theviral genome and are capable of producing infectious virus but for thedeletion of the packaging signal located at the left end of thewild-type viral genome. The second component of that system comprises aseries of “shuttle” plasmids that contain the left approximately 340 ntof the Ad genome including the packaging signal, optionally apolycloning site, or optionally an expression cassette, followed byviral sequences from near the right end of E1 to approximately 15 mu oroptionally to a point further rightward in the genome. The viralsequences rightward of E1 overlap with sequences in the pBHG plasmidsand, via homologous recombination in cotransfected host cells, produceinfectious virus. The resulting viruses contain the packaging signalderived from the shuttle plasmid, as well as any sequences, such as aforeign DNA inserted into the polycloning site or expression cassettelocated in the shuttle plasmid between the packaging signal and theoverlap sequences. Because neither plasmid alone has the capability toproduce replicating virus, infectious viral vector progeny can onlyarise as a result of recombination within the cotransfected host cell.However, as has been noted above, such homologous recombinationprocesses can be inefficient, resulting in variable success in theisolation of vectors and occasional failure, particularly in the handsof those who are not specifically skilled in the art of virology, andmore particularly, in the art of adenovirology.

Site-specific recombination catalysed by an efficient recombinase, suchas the Cre or FLP recombinase, can be many fold more efficient thanhomologous recombination. This invention disclosure provides methods andnucleic acid constructs which significantly enhance the ease ofproduction of viral vectors by the two plasmid method by enablingsite-specific recombination between individual nucleic acids constructs,neither of which alone is able to replicate and produce infectiousadenovirus. The methodology described herein furthermore utilizesCre-loxP and other known recombination systems for efficientintroduction of mutations of viral genes into the viral genome.Furthermore, the instant methodology is also applicable to insertion offoreign DNA sequences into various regions of the viral DNA, in additionto the E1 region classically used for that purpose. In additionalembodiments of this invention, site-specific recombination is utilizedin combination with infectious viral DNA having covalently boundterminal protein (DNA-TP complex), at either or both 5′ strands of theDNA (i.e. either the coding or non-coding strand). Additionalembodiments and applications of the site-specific recombinationmethodology will also become apparent based on the instant disclosure.

Having generally described the purposes, advantages, applications andmethodology of this invention, the following specific examples areprovided to describe in a detailed fashion, various embodiments of thisinvention. However, it should be appreciated that the inventiondescribed herein is not limited to the specifics of the followingexamples, which are provided merely as a guide for those wishing topractice this invention. The scope of the invention is to be evaluatedwith reference to the complete disclosure and the claims appendedhereto.

The following examples using the human adenovirus serotype 5 are notmeant to be limiting. One skilled in the art would realize that similarplasmids, viruses and techniques could be utilized with a differenthuman adenovirus serotype, for example Ad2. Similarly, the use of humanAds is not meant to be limiting since similar plasmids, viruses andtechniques could be utilized for different non-human adenoviruses, forexample bovine. Similarly, the use of adenoviruses is not meant to belimiting since similar plasmids, viruses and techniques could beutilized with other viruses, both human and non-human, for examplebaculovirus.

Use of Cre recombinase in these and other examples is not meant to belimiting as a person skilled in the art will readily appreciate thatother enzymes capable of catalysing site-specific recombination betweenDNA sequences recognized by said enzymes could equally be employed inplace of the Cre recombinase. An example, not meant to be limiting, ofsuch an enzyme that could be substituted for Cre is the “FLP”recombinase of yeast in combination with its target site FRT (O'Gormanet al. Science 251, 1351, 1991).

A component of the invention is the use of human cells, such as 293cells or other cells that may be deemed suitable in that they supportthe replication of the viral components of the invention, that expressCre recombinase and that can be transfected with the plasmids describedherein and in the examples which follow, to generate a virus containingthe desired modifications such as an insertion of foreign DNA or amodified fibre gene. It will be appreciated by those skilled in the artthat the requisite cell lines can be generated by transfecting 293 cellsor other cells, with a plasmid comprising the coding sequences for Creunder the control of suitable regulatory sequences, including a promoterand polyadenylation signal and containing, in addition, a selectablegene encoding, for example, resistance to G418 or histidinol. A personskilled in the art can readily obtain drug resistant cells that expressthe Cre recombinase in addition to the drug resistance gene used forselection. It will also be appreciated by one skilled in the art, basedon the present disclosure, that host cells can also be induced totransiently express a recombinase by transfection with a plasmidcomprising an expression cassette containing said recombinase gene or byinfection with a viral vector that expresses the recombinase. Thus theexample of 293Cre cells or other permanently transformed recombinaseexpressing cell lines is not meant to be limiting.

EXAMPLE 1 Two-Plasmid, Site-Specific Adenoviral Recombination

FIG. 1 provides a graphic representation of the use of a plasmid,pBHG10lox, which comprises a circularized form of the Ad genome in whichpart or all of the E1 region, including the packaging signal, issubstituted by sequences comprising a bacterial plasmid origin ofreplication and an antibiotic resistance gene, such as that encodingampicillin resistance. The plasmid further comprises a loxP site nearthe 5′ end of the pix gene of the Ad genome. The plasmid may also,optionally, have a deletion of E3 sequences (as shown in thisillustration by the symbol Δ3) which may optionally be substituted withone or more unique cloning sites for insertion of foreign DNA in the ΔE3region.

A second component of the invention comprises a “shuttle plasmid”containing an ITR of the virus genome and a packaging signal, apolycloning site into which may be inserted a foreign DNA such as thatencoding for bacterial β-galactosidase (LacZ) or any other gene,expression of which is desired either in a gene therapeutic or vaccinecontext, and a loxP site inserted in the same relative orientation asthe loxP site in pBHG10lox. To obtain high-efficiency rescue of theforeign DNA into an infectious viral vector, the two plasmids arecotransfected into human cells, such as 293Cre cells, PER-C6 cells, 911cells, and the like, engineered to express Cre and which, in addition,express the E1 region of the Ad genome. It should be appreciated thatthe manner of provision of the recombinase is not critical. Therecombinase may be constitutively expressed by the cell into which thetwo plasmids are introduced. The recombinase may be provided in trans,via a third plasmid, or in cis, by inclusion of a recombinase expressioncassette in one of the introduced plasmids. In addition, it will beappreciated that any recombinase which efficiently induces site-specificrecombination between sequences present on the two plasmids may beemployed according to this methodology. Thus, the FLP recombinase, whichrecognizes the sequences known as FRT, may be used in place of theCre/loxP combination, and thus, wherever Cre or loxP are mentionedherein, such mention should be read to include any other site-specificrecombination system now known or henceforth discovered, when applied tothe specific techniques disclosed and claimed herein.

Cre-mediated recombination results in formation of joint molecules thatgenerate infectious viruses containing the foreign DNA insert. BecausepBHG10lox lacks the viral packaging signal, the only viruses that canform are those containing the packaging signal and foreign DNA of theshuttle plasmid. These are generated in large numbers because of thehigh-efficiency and specificity of Cre recombinase, and there is nobackground of non-recombinant virus which, according to a method such asthat of Hardy et al., J. Virol. 71(3):1842-1849, (1997), even afterthree sequential passages in Cre expressing cells, results in a vectorpreparation still contaminated by starting (non-recombinant) virus.

EXAMPLE 2 Comparison of Homologous and Site-Specific Recombination

FIG. 2 illustrates use of a modified shuttle plasmid wherein Adsequences from about 10 mu to about 15 mu are present to the right ofthe lox site. These sequences permit homologous recombination to occurin the absence or presence of Cre. A shuttle plasmid such as that shownin this figure is generally used only for comparison purposes to assessthe relative efficiency of homologous versus Cre-mediate recombination.As will be seen in the subsequent description of the invention, in thepresence of Cre, overlapping sequences are unnecessary and can beomitted, although this disclosure does not require the absence of suchsequences.

EXAMPLE 3 Sequences Useful in the Production of Plasmids which may beRecombined in a Site-Specific Manner to Produce Adenoviral Vectors

FIG. 3 illustrates sets of oligonucleotides used in various cloningprocedures. The double stranded oligonucleotide (SEQ ID NO:1 and SEQ IDNO: 2; AB3233/3234) contains a loxP site with restriction sites for ScaIand EcoRI at one end of the oligo outside of the loxP region. Whenannealed, the oligonucleotides have BamHI/Bgl II overhangs which aredesigned for cloning into and concomitant destruction of the BglII site.The internal ScaI site found in (SEQ ID NO:1 and SEQ ID NO: 2;AB3233/3234) was designed to facilitate determination of the orientationof the linker and also for subsequent deletion of Ad5 sequences fromm.u. 9.8-15.8. The second linker (SEQ ID NO:3 and SEQ ID NO:4;AB14626/14627) has EcoRI and SalI overhangs and a multiple cloningregion containing SmaI, BglII, HindIII and ScaI restriction sites.

EXAMPLE 4 Construction of Bacterial Plasmids Containing CircularizedForms of the Adenovirus Genome Suitable for Rescue of Viral VectorsUsing Site-Specific Recombination According to the General SchemeIllustrated According to FIG. 1

FIG. 4A illustrates production of a plasmid, pBHG10lox, a derivative ofpBHG10, modified to contain a loxP site at the 3′ end of the E1deletion. As can be seen with reference to the figure, this plasmid wasconstructed by replacing the 4604 bp Bst1107I fragment from pBHG10 withthe 2326 bp EcoRV/Bst1107I fragment from pE1sp1Alox. The plasmidpΔE1sp1Alox (FIG. 5) was constructed by inserting an oligonucleotidebearing a loxP site (comprised of annealed oligos (SEQ ID NO:1 and SEQID NO: 2; AB3233/3234) into the BglII site of pΔE1sp1A. Foreignsequences can be inserted into the unique PacI site of pBHG10lox forrescue of genes in E3. The plasmid illustrated in FIG. 4 can be selectedfrom the series pBHG10 (as illustrated), pBHG11, pBHGE3, or likeplasmid, for modification to contain a lox P site near the 3′ end of E1ie. near the 5′ end of the pIX gene at approximately nt 3520 in theconventional sequence of Ad5. Optionally E1 sequences from approximatelynt 188 to approximately 3520 may be deleted from said plasmid. Like theparental plasmid (such as pBHG10, pBHG11 or pBHGE3) the modified pBHGderivative (eg. pBHG10lox, pBHG11lox, pBHGE3lox, or like plasmid) lacksthe packaging signal (2), and is consequently incapable of producinginfectious virus in transfected host cells.

FIGS. 4B-1 and 4B-2 illustrate the construction of a plasmid,pBHGdX1Plox, containing a modified E3 deletion (taken from pFG23dX1) anda lox site 5′ of the pIX gene. The plasmid pFG23dX1P was constructed byinserting an oligonucleotide containing a PacI site (AB14566;5′-CTAGCTTAATTAAG-3′, SEQ ID NO.:9) into the XbaI site of pFG23dX1. Theplasmid pNG17 was constructed by cloning the 6724 bp SpeI/ClaI fragmentfrom pBHG10lox into pBluescript. The plasmid pNG17dX1P was constructedby replacing the 1354 bp SpeI/NdeI fragment from pNG17 with the 2129 bpSpeI/NdeI fragment from pFG23dX1P. The plasmid pBHGdX1P was constructedby replacing the 6724 bp SpeI/ClaI fragment from pBHG10lox with the 7495bp SpeI/ClaI fragment from pNG17dX1P.

FIG. 4C illustrates the construction of a plasmid containing a wild-typeE3 region and a loxP site 5′ of the pIX gene. The plasmid pBHGE3lox wasconstructed by replacing the 6724 bp SpeI/ClaI fragment from pBHG10loxwith the 9377 bp SpeI/ClaI fragment from pBHGE3.

EXAMPLE 5 Construction of Shuttle Plasmids for Recombination withAdenoviral Rescue Plasmids Constructed According to Example 4

As described above, a second embodiment of the invention comprises ashuttle plasmid selected from a series of plasmids containing,minimally: the left end of the viral genome including all or most of theleft Inverted Terminal Repeat (ITR nts 1-103 of the Ad 5 DNA) and thepackaging sequence, and optionally a polycloning site or optionally anexpression cassette. With reference to FIGS. 5-8, such shuttle plasmidsare modified to contain a lox P site in the same orientation as the loxPsite in the pBHG derivative, (see Example 4, referred to herein as the“rescue plasmid”), said loxP site being positioned in said shuttleplasmid to the right of said polycloning site or said expressioncassette.

FIG. 5 illustrates the construction of shuttle plasmids derived frompΔE1SP1A and pΔE1SP1B wherein loxP sites are introduced 5′ of the pIXgene. The plasmids, pΔE1sp1A and pΔE1SP1B are left end shuttle plasmidscontaining Ad5 sequences from m.u. 0-15.8 with E1 sequences deletedbetween m.u. 1 and 9.8. They are identical except that the restrictionsites in the multiple cloning region are reversed. A synthetic loxPlinker (SEQ ID NO:1 and SEQ ID NO:2; AB3233/3234) was introduced intothe BglII site of each plasmid generating pΔE1SP1Alox and pΔE1SP1Blox.Ad5 sequences from m.u. 9.8-15.8 were removed by digesting the plasmidswith NruI, partially cutting with ScaI followed by self-ligation. Theplasmids thus generated are called pΔE1SP1AloxΔ and pΔE1SP1BloxΔ.

FIG. 6A illustrates the construction of pMH4lox and pMH4loxΔ plasmidsthat contain a promoter and polyadenylation signal and polycloning sitesfor insertion of foreign DNA to produce expression cassettes in whichtranscription is driven by the murine cytomegalovirus immediate earlygene promoter. Plasmid pVDB3 is derived from pMH4 but contains apUC-based origin of replication, rather than a pBR322 origin. Itcontains Ad5 sequences from m.u. 0-15.8 with E1 sequences deletedbetween m.u. 1 and 9.8 and substituted with an expression cassette: a0.5 kbp (−491 to +36) fragment of the MCMV IE promoter, uniquerestriction enzyme sites for cloning (Eco RI, Nhe I, Bam HI and Sal I)followed by an SV40 polyadenylation signal. To make pMH4lox, a loxPlinker (SEQ ID NO:1 and SEQ ID NO:2; AB3233/3234) was introduced intothe BglII site of pVDB3. Ad5 sequences m.u. 9.8-15.8 were deleted frompMH4lox by digesting with Hind III, treating with the Klenow fragment ofE. coli DNA polymerase then partially digesting with Sca I followed byself-ligation. The resulting shuttle plasmid, pMH4loxΔ, can be used withpBHG10lox to produce Ad vectors via Cre/lox mediated recombination. Tomake pMH4loxΔ a more flexible plasmid for cloning purposes, a linker(SEQ ID NO:3 and SEQ ID NO:4; AB14626/14627), containing a differentmultiple cloning region, was introduced between the Eco RI and Sal Isites resulting in pMH4loxΔlink.

FIG. 6B illustrates the construction of plasmid pVDB3. A PvuI to Bst11071 fragment from pMH4 (Microbix Biosystems) was ligated to a Bst11071 to Pvu I fragment from pD47E1 containing a pUC-based (pNEB193, NewEngland Biolabs) origin of plasmid DNA replication to generate pVDB3.

FIG. 7 illustrates construction of HCMV loxP plasmids in whichtranscription of foreign genes is regulated by the human cytomegalovirusimmediate early gene promoter. The plasmids pCA13 and pCA14 contain theAd5 genomic sequences from m.u. 0 to 15.8 with E1 sequences between m.u.1 and 9.8 replaced by the HCMV IE promoter (−299 to +72, relative to thetranscription start), a polycloning region and an SV40 polyadenylationsignal. (Plasmids pCA13 and pCA14 are available from MicrobixBiosystems). The expression cassette in each case is oriented parallelto the direction of E1 transcription (rightwards). The only differencebetween pCA13 and pCA14 is in the orientation of the multiple cloningregion. The plasmids pCA13(ΔBglII) and pCA14(ΔBglII) were generated bydigesting pCA13 and pCA14 partially with BglII, Klenowing andself-ligating. A synthetic loxP oligonucleotide (SEQ ID NO:1 and SEQ IDNO:2; AB3233/3234) was introduced into the unique BglII sites ofpCA13(ΔBglII) and pCA14(ΔBglII) producing pCA13lox and pCA14loxrespectively. Ad5 sequences, m.u. 9.8-15.8, were removed from pCA13loxand pCA14lox by cutting each plasmid with NruI and partially digestingeach with ScaI followed by self ligation. The resulting plasmids,pCA13loxΔ and pCA14loxΔ are useful shuttle plasmids for the rescue offirst generation Ad vectors by Cre/lox recombination.

FIG. 8A illustrates the construction of a plasmid, pCA36loxΔ, for rescueof the β-galactosidase gene into adenovirus vectors. Naturally, therescued gene may be any foreign gene, and is not restricted to the useof a marker gene, such as the marker beta-gal gene, which is used hereinfor illustrative purposes. The plasmid pCA36 contains the β-gal cDNAunder control of the short MCMV IE promoter (−491 to +36) followed by anSV40 polyadenylation signal. Plasmid pCA36 was made by inserting theLacZ gene into pMH4 (available from Microbix Biosystems) and isdescribed by Addison, C. L., Hitt, M., Kunsken, D. and Graham, F. L., in“Comparison of the human versus murine cytomegalovirus immediate earlygene promoters for transgene expression in adenoviral vectors,” J. Gen.Virol. 78: 1653-1661, 1997.” A synthetic loxP site (SEQ ID NO:1 and SEQID NO:2; AB3233/3234) was introduced into the Bgl II site of pCA36resulting in pCA36lox. This plasmid was then digested with Nru I andpartially digested with Sca I, a 7646 bp fragment was gel purified andself ligated yielding pCA36loxΔ. This plasmid contains Ad sequences fromm.u. 0-1, and not only has the deletion of E1 sequences present in theparental plasmids pCA36 and pCA36lox, but additionally is deleted of Ad5sequences from m.u. 9.8-15.8.

EXAMPLE 6 Demonstration of Enhanced Efficiency of Site-SpecificRecombination in Comparison with Homologous Recombination

In a third embodiment of the invention, two plasmids containing loxP orother recombinase recognition sites are cotransfected into 293Cre orother appropriate cells (expressing an appropriate recombinase, Cre forpurposes of this example). The Cre enzyme catalyses site-specificrecombination between said lox P sites present in each vector. Asillustrated in FIG. 1, it will be readily seen by one skilled in the artthat Cre-mediated recombination between said lox P sites generates aviable virus by joining pBHG sequences to a DNA segment containing 2 andITR sequences. Furthermore, by virtue of the design and construction ofthe pBHG derivative and the shuttle plasmid, the resulting viral vectorcontains the expression cassette located to the left of the lox P sitein said shuttle plasmid, thereby providing a simple and efficient meansfor isolating viral vectors containing foreign DNA insertions andexpression cassettes for synthesis of proteins from foreign genes.

To test and demonstrate the validity of the approaches outlined aboveand to determine the degree of improvement in efficiency of vectorisolation compared to known methods, a number of experiments wereconducted in which a vector carrying a LacZ expression cassette insertednear the left end of the Ad genome was constructed. The efficiency ofCre/lox mediated recombination was compared with that of homologousrecombination, by measuring the numbers of virus plaques obtained fromcotransfections of 293 cells versus the numbers obtained followingcotransfections of 293Cre4 cells (see, for example, U.S. patentapplication Ser. No. 08/473,168, filed Jun. 7, 1995; see alsoWO96/40955, hereby incorporated by reference).

The results shown in Table 1 indicate that Cre/lox mediatedrecombination (cotransfections of 293Cre4 cells with plasmids that bothcontain lox sites) was approximately 35-fold more efficient thanhomologous recombination (cotransfections of 293 cells orcotransfections of 293Cre4 cells with plasmids that do not both containlox sites). A 35-fold increase represents a very significant andunexpectedly high improvement over efficiencies of vector rescue whenvirus isolation is dependent on homologous recombination. Coupled withthe fact that the only infectious virus present in the transfected cellpreparation are recombinants, rather than contaminating starting virus,the efficiency, cleanliness and convenience of this method in comparisonto known methods represent significant advances in the art. Thus, withthis new method it will be possible to reduce the amount of plasmid DNAused in cotransfections and reduce the number of dishes of 293 (293Cre)cells needed in cotransfections for rescue of viral vectors. It willalso aid in the rescue of constructs which, for unknown reasons, mightbe otherwise difficult to rescue (e.g. rescue of vectors containinglarge foreign DNA inserts in E1 is often inefficient for reasons thatare not known).

To confirm that the enhanced efficiency of plaque formation followingcotransfection of 293Cre cells with pCA36+pBHG10lox was due to Cre-loxdependent recombination (versus, for example, enhanced efficiency ofhomologous recombination) we constructed a derivative of pCA36lox, namedpCA36lox (see FIG. 8A), from which overlapping Ad sequences to the rightof the lox site had been removed, thus eliminating any possibility ofhomologous recombination. This new shuttle plasmid was then tested forability to generate vectors in a second experiment in which 293 or293Cre cells were cotransfected with this plasmid or with pCA36 orpCA36lox for comparison along with pBHG10lox. It can be seen from theresults shown in Table 2 that pCA36loxΔ only generated viral plaquesfollowing cotransfection of 293Cre cells with pBHG10lox. In contrastpCA36 or pCA36lox were able to generate small numbers of plaques on 293cells. However, again, the efficiency was markedly enhanced if 293Crecells were cotransfected with pCA36lox and pBHG10lox. Thus the use ofCre-lox recombination results in a surprisingly efficient system forrescue of foreign DNA into Adenovirus vectors.

To confirm that transfection of 293Cre cells with pCA36lox (alacZ-containing shuttle plasmid with a loxP site located between theexpression cassette and the pIX coding sequence as illustrated in FIG.8) and pBHG10lox resulted in viruses containing the desired insert offoreign DNA, 26 recombinant plaques were isolated, expanded and analysedfor expression of LacZ. All 26 (100%) were positive for β-galactosidaseexpression. Furthermore, analysis of the structure of the virusesconfirmed that all 26 had the expected DNA structure illustrated inFIG. 1. Further confirmation of the efficiency and specificity of theCre/lox system for rescue of expression cassettes was obtained throughanalysis of 6 plaque isolates obtained by cotransfection of 293Cre cellswith pCA36loxΔ and pBHG10lox (Table 2). All 6 plaque isolates expressedβ-galactosidase and all 6 had the expected DNA structure illustrated inFIG. 1. Because 100% of recombinant viruses produced by cotransfectionof 293Cre cells with plasmids containing appropriately engineered loxsites have the correct structure and express the transgene,(β-galactosidase in these examples), it will be appreciated by thoseskilled in the art that one could readily produce recombinant virusescarrying other foreign DNA inserts by constructing shuttle plasmidsderived from the plasmids shown in FIGS. 5, 6 and 7 or similar plasmids,and cotransfecting said modified shuttle plasmids into 293Cre or likecells, along with pBHG10lox or similar pBHG plasmids containing a loxsite near the end of E1. It will be further appreciated by those skilledin the art that because of the high-efficiency of rescue with thisapproach, only small numbers of 293Cre cultures and small amounts of DNAneed be used to obtain the desired recombinant viruses. Furthermore,because only the desired recombinant viruses are obtained from saidcotransfections, it would not be essential to plaque purify and analyseviral progeny obtained according to the method of this invention. Inaddition, after the initial isolation of the recombinant viruses from293 Cre cells, said viruses can be propagated in host cells such as 293,911 or PERC-6 cells or the like which do not express recombinase.

EXAMPLE 7 Site-Specific Shuttle Plasmid-Virus Recombination

Hardy et al., J. Virol. 1997, March: 71(3):1842-1849, and see alsoWO97/32481 disclosed a method whereby an infectious DNA vector was usedin combination with a plasmid in combination with lox-Cre recombinationto generate recombinant adenoviruses. However, according to that method,residual infectious starter virus remain in the recombinant viruspreparation, requiring repeated passage of the preparation in a Creexpressing cell to eliminate this background. An advancement to suchtechniques is provided herein by combination of Cre-lox recombinationand use of adenoviral DNA bound to the adenoviral terminal protein (TP).The result of this combination is high-efficiency infection combinedwith site-specific recombination.

The use of a two plasmid system for isolation of viral vectors ormodified viruses is not meant to be limiting. From the instantdisclosure, it will be appreciated by those skilled in the art that onecould use, as one component of the system, viral DNA from a modifiedvirus whose genome contains lox P sites at useful positions. Anexcellent example, not meant to be limiting, is use of AdLC8, AdLC8c orAdLC8cluc described by Parks, R. J., Chen, L., Anton, M., Sankar, U.,Rudnicki, M. A. and Graham, F. L., in “A new helper-dependent adenovirusvector system: removal of helper virus by Cre-mediated excision of theviral packaging signal,” Proc. Natl. Acad. Sci. U.S. 93: 13565-13570,1996. These viruses contain a “floxed” packaging signal, which isexcised following virus infection of 293Cre cells. Therefore,cotransfection of 293Cre cells with viral DNA extracted from AdLC8,AdLC8c or AdLC8cluc in such a way as to retain the covalent linkage toTP, according to methods taught by Sharp et al., “The infectivity ofadenovirus 5 DNA-protein complex,” Virology, 1976 December:75(2):442-456; Chinnadurai, et al., “Enhanced infectivity of adenovirustype 2 DNA and a DNA-protein complex,” J. Virol. 1978 April:26(1):195-199, and a shuttle plasmid such as that illustrated in FIGS.5, 6, 7 or 8A results in Cre-mediated recombination to generate a newvector containing the sequences derived from the shuttle plasmid,spanning the region from the ITR and packaging signal of the shuttleacross the optional polycloning site or optional expression cassette tothe lox P site of said shuttle plasmid. For example, not meant to belimiting, as illustrated in FIG. 8B, using a lacZ-encoding plasmid,similar to that shown in FIG. 8A, and AdLC8c DNA-TP, one skilled in theart could readily isolate the desired recombinant virus containing lacZor other foreign genes by cotransfection of 293Cre cells with DNAextracted from AdLC8c-TP and said Lac Z-encoding plasmid. Optionally, asillustrated in FIG. 8C, one could cotransfect 293Cre cells withrestriction endonuclease treated AdLC8c DNA-TP and a shuttle plasmidselected from the set of plasmids illustrated in FIGS. 5, 6, 7 and 8A toproduce infectious virus by Cre-mediated recombination. The viral DNAextracted from AdLC8c according to this method retains the terminalprotein which has been shown to increase the efficiency of transductionof recipient cells with said DNA (Sharp P A, Moore C, Haverty J L, “Theinfectivity of adenovirus 5 DNA-protein complex,” Virology 1976December; 75(2):442-456). It will be apparent to those skilled in theart that the left most lox site is not needed and may optionally bedeleted if AdLC8cDNA-TP is to be cut with restriction enzymes priorcotransfection. Furthermore, optionally, after restriction enzymedigestion, the large right end fragment of AdLC8cDNA-TP could bepurified prior to cotransfection.

FIG. 8D is a diagrammatic representation of a method for constructingshuttle plasmids expressing Cre. The Cre expression cassette wasobtained from the plasmid pLC2 (Chen, L., Anton, M. and Graham, F. L.,“Production and characterization of human 293 cell lines expressing thesite-specific recombinase Cre,” Somat. Cell and Molec. Genet.22:477-488, 1996), as a 2175 bp BglII fragment which was end-modifiedwith Klenow DNA polymerase and inserted into the EheI site of pCA36loxΔto generate pCA36loxΔCreR and pCA36loxΔCreT. The plasmid pCA36loxΔCreITRwas constructed by replacing the 1402 bp ScaI/KpnI fragment inpCA36loxΔCreT with the 2753 bp ScaI/KpnI fragment from the plasmidpRP1029. Plasmid pCA36loxΔCreITR contains ITR junctions which are knownto be functionally capable of generating replicating linear Ad DNAmolecules (Graham, F. L., “Covalently closed circles of human adenovirusDNA are infections,” The EMBO J. 3, 2917-2922, 1984).

FIG. 8E provides a schematic representation of a cotransfectionexperiment wherein a pBHG10lox plasmid and a “Lox” shuttle plasmidexpressing Cre are introduced into 293 cells in order to generate Adexpression vectors, without having to use cells which stably expressCre. This technique is applicable to any cell type suitable for Advector generation, including but not limited to 293 cells, and PER-C6cells (Fallaux et al., Hum. Gene Ther. 1998, Sep. 1; 9(13):1909-17), 911cells (Fallaux et al., Hum. Gene Ther. 1996 Jan. 20; 7(2):215-222), orother cells. A shuttle plasmid such as pCA36loxΔCreITR of FIG. 8D isalso suitable for generation of an Ad vector. The efficiency of Advector rescue by cotransfection with pBHGlox and various shuttleplasmids is summarized in Tables 3 and 4. It can be seen from theresults in Table 4 that inclusion of an ITR junction in the shuttleplasmid increases the efficiency of rescue significantly. Thus,provision of an ITR junction is a preferred, although not required,embodiment.

Insertion of an expression cassette encoding Cre recombinase in theshuttle plasmid is not meant to be limiting as one skilled in the artwill appreciate that one could also insert a Cre cassette in the largerplasmid, pBHG10lox. An example, not meant to be limiting, is diagrammedin FIG. 8F, which illustrates the construction of such a plasmid. Itwill be appreciated that the Cre expression cassette could be carried byeither of the two plasmids used in the cotransfections such as thatillustrated in FIG. 1, or by both of them, so that Cre is supplied atadequate levels in cotransfected 293 cells to catalyse efficientrecombination between the lox sites of the cotransfected plasmids. Thusmention of the use of 293Cre cells or like cells expressing Crerecombinase is not meant to be limiting.

FIG. 8F demonstrates the construction of an Ad genomic plasmid encodingCre. The plasmid pBHG10loxΔ was constructed by collapsing pBHG10lox withSpeI and PshAI. The Cre expression cassette, taken from the plasmid pLC2as a 2175 bp BglII fragment, was inserted into the BamHI site ofpBHG10loxΔ to generate pBHG10loxΔCreR and pBHG10loxΔCreT. The 1238 bpBst1107I/PacI fragment from pBHG10loxΔCreR and pBHG10loxΔCreT wasreplaced with the 22380 bp Bst1107I/PacI fragment from pBHG10lox togenerate pBHG10loxCreR and pBHG10loxCreT, respectively.

EXAMPLE 8 Rescue of Foreign DNA and Mutations into Any Desired Locationin the Adenoviral Genome

The above examples illustrating rescue of foreign DNA into the E1 regionof Ad vectors are not meant to be limiting. It will be appreciated bythose skilled in the art that one could equally follow the instructionsoutlined above to construct similar plasmids for the rescue ofinsertions or mutations or deletions into E1 or other regions of theviral genome. For example, not meant to be limiting, one could constructa series of analogous plasmids suitable for rescue of fibre mutationsinto the viral genome or for rescue of foreign DNA inserts in the E3region of the viral genome into infectious virus. An example, not meantto be limiting, is provided in FIG. 9A, which is a diagrammaticrepresentation of a method for rescuing fibre mutations into infectiousvirus using Cre-loxP recombination. Cotransfection of 293Cre cells withpFG173lox and a shuttle plasmid containing a loxP site 5′ of the fibregene results in site-specific recombination between the lox sites andrescue into infectious virus of the adenoviral sequences of the shuttle,which sequences may optionally contain a mutated fibre gene.

FIG. 9B is a diagrammatic representation of a method for constructing aplasmid containing a lox site and ampicillin resistance genesubstituting for the fibre gene. Starting with a plasmid such aspAB14lox, construction of which is described in FIG. 14, the DNAsequences between the Cla I site and the Blp I site containing fibre aresubstituted with a DNA segment containing the ampicillin resistance geneand a plasmid origin of DNA replication (which may optionally beobtained by restriction endonuclease digestion of an ampicillinresistant plasmid such as pCA14 (Microbix Biosystems)).

FIG. 9C is a diagrammatic representation of a method for combining theplasmid of FIG. 9B with pFG173 to produce pFG173lox for rescuing fibremutations into infectious virus using Cre-lox recombination. The plasmidpAB14lox illustrated in FIG. 9B comprises Ad sequences 3′ of fibre to mu100. The plasmid additionally contains viral DNA sequences 5′ of fibre,but has all of the fibre coding sequences deleted and substituted with aplasmid origin of DNA replication and an antibiotic resistance gene,such as for ampicillin resistance. Sequences from pAB14loxΔ can berecombined with pFG173 (Microbix Biosystems) by homologous recombinationin E. coli (Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A,Mehtali M., “Efficient generation of recombinant adenovirus vectors byhomologous recombination in Escherichia coli,” J Virol 1996 July;70(7):4805-4810). The resulting plasmid, pFG173lox, has a deletion ofsequences comprising all of the fibre gene or optionally part of thefibre gene or optionally all or part of E4 or optionally a deletion ofall or part of both fibre and E4, and is consequently unable to produceinfectious virus following transfection of cells. However, onrecombination with a plasmid such as pFG23dX1lox or a similar plasmid,infectious virus can be readily generated, as illustrated in FIG. 9A.Said recombination can be efficiently catalysed by Cre recombinase, ifpFG173lox and pFG23dX1lox are cotransfected into 293Cre cells or similarhost cells expressing Cre recombinase.

Construction of plasmids suitable for rescue of fibre or E4 genemutations or deletions or substitutions can be readily accomplished byone skilled in the art based on the present disclosure. An example, notmeant to be limiting, of the construction of one such plasmid isillustrated in FIG. 10, which is a diagrammatic representation of aplasmid containing the right approximately 40% of the virus genome,wherein a lox P site has been inserted near the 5′ end of the fibregene. PFG23dX1 contains the right approximately 40% of the Ad 5 genomefrom nt 21563 (mu 60) to approximately the right end of the genome (mu100) cloned into the BamHI site of pBR322 and additionally has adeletion of Ad5 sequences from 28593 to 30471, comprising most of E3(Haj-Ahmad, Y. and Graham, F. L., “Development of a helper independenthuman adenovirus vector and its use in the transfer of the HerpesSimplex Virus thymidine kinase gene,” J. Virol. 57, 267-274, 1986).PFG23dX1 was digested with XbaI and a synthetic oligonucleotide (SEQ IDNO:5 and SEQ ID NO:6; AB6920/AB6921, FIG. 3) containing a loxP site wasinserted. The resulting plasmid, pFG23dX1lox, can be used for generationof infectious virus by cotransfection of 293Cre cells with a plasmidsuch as pFG173lox (FIG. 9A). Optionally, viral genes, such as thoseencoding fibre or genes of E4 can be mutated in pFG23dX1lox and theresulting mutations rescued into virus. Because Ad sequences 5′ of thelox site (counterclockwise in the diagram) are not necessary whenCre-mediated site specific, rather than homologous, recombination isused to generate infectious virus, viral sequences between a unique Bst11071 site and a BsiW1 site immediately 5′ of the lox P site weredeleted to generate pFG23dX1loxc.

One skilled in the art would appreciate, based on the instantdisclosure, that just as Cre recombinase may be provided by inserting aCre expression cassette in one or another or both of the cotransfectingplasmids to facilitate recombination between plasmids designed to rescuemutations or insertions in E1, similarly, one may insert said expressioncassette into either or both of the plasmids to be recombined asdiagrammed in FIG. 9A so that site specific recombination can beachieved in host cells that do not express the recombinaseconstitutively. In a preferred embodiment, the shuttle plasmid thusmodified would be further modified to contain a junction of ITRs as theresults shown in Table 4 indicate that said junction results in asignificant improvement in efficiency of virus production. As in theexamples illustrated in FIGS. 8D and 8F, said plasmids would most oftenbe designed so that the Cre expression cassette would not be rescuedinto the infectious viral genomes that are thus generated.

Examples illustrating rescue of mutations into infectious virus are notmeant to be limiting as one skilled in the art could readily appreciatethat the methods described herein are equally employed to rescueinsertions of foreign DNA into the viral genome. An example of asuitable plasmid that is readily constructed is pFG23dX1LacZlox. FIG. 11is a diagrammatic representation of said plasmid wherein a foreign DNA,such as a gene encoding bacterial lacZ, is inserted between the lox Psite and the fibre gene. In this example, not meant to be limiting, anexpression cassette encoding β-galactosidase is inserted into the Cla Isite adjacent to the loxP of pFG23dX1lox (FIG. 10) for subsequent rescueinto infectious virus by the method illustrated in FIG. 9A. It will beappreciated by those skilled in the art that other foreign DNAs couldreadily be rescued into infectious virus genomes by the methodsillustrated above. Said foreign DNA segment could be a separateexpression cassette or a fusion of sequences encoding peptide sequencesto sequences encoding fibre, said peptide sequences representing, forexample, a ligand to a cell surface receptor such that the rescued virusexpressing a modified fibre would have novel and useful cell attachmentproperties. This example is not meant to be limiting as it will beappreciated by one skilled in the art that lox P sites can readily beintroduced into other positions of the viral DNA for substitution ofother virion genes with mutated counterparts.

These examples are not meant to be limiting as one could construct aplasmid similar to pFG173lox from which other viral genes have beendeleted such as, for example, those of E1 such that the resultingviruses generated by Cre-mediated recombination are E1 deleted viruses.

EXAMPLE 9 Use of Engineered Adenoviruses Produced According to thisInvention

The use of the two plasmid system in combination with Cre-mediatedsite-specific recombination is not meant to be limiting as one skilledin the art will readily appreciate that, as taught for the generation ofviruses carrying E1 mutations, deletions and insertions, one couldemploy viral DNA isolated from suitably engineered viruses for themanipulation of the viral genome by Cre-mediated recombination. Forexample, as illustrated in FIGS. 12 and 13, 293Cre cells arecotransfected with DNA extracted from a virus containing a floxed fibregene in such a way as to retain either or both terminal proteins, TP.Optionally the DNA is digested with restriction enzymes that cutsequences between the lox sites prior to cotransfections. It will beapparent to those skilled in the art, based on the instant disclosure,that the right most lox site is not needed and may optionally be deletedor omitted if DNA-TP is to be cut with restriction enzymes prior tocotransfection. As with the two plasmid method, the method of FIGS. 12and 13 is employed to rescue mutations in the fibre gene or in E4 or torescue foreign DNA inserts as in FIG. 13.

To confirm that it is possible to insert into the adenovirus genome loxsites that flank a gene such as that encoding fibre, the plasmid shownin FIG. 14, called pAB14flox, was constructed. This plasmid contains alox site inserted into the unique Blp I site in pAB14, which is locatedbetween the 3′ terminus of the fibre gene and the coding regions of E4genes. A second lox site was inserted into the XbaI site upstream offibre. PAB14flox (fibre flanked by lox sites) was rescued intoinfectious virus by cotransfection with pFG173 (described in Hanke, T.,Graham, F. L., V. Lulitanond and D. C. Johnson, “Herpes simplex virusIgG Fc receptors induced using recombinant adenovirus vectors expressingglycoproteins E and I,” Virology 177: 437-444, 1990. PFG173 is availablefrom Microbix Biosystems) as illustrated in FIG. 15, to produceAdfloxfibre. In two experiments, 293 cells were cotransfected withpAB14flox and pFG173, and two plaque isolates were obtained in eachexperiment (from 8 cotransfected dishes of 293 cells in experiment 1,and from 4 dishes in experiment 2). Two plaques were expanded andanalysed and shown to have the expected DNA structure as illustrated inFIG. 15.

Upon transfection of 293Cre cells with DNA-TP complex of an Ad virus,such as Adfloxfibre-TP depicted in FIG. 15, said floxed fibre gene isexcised by site-specific recombination between similarly oriented lox Psites, resulting in noninfectious viral DNA (as fibre is an essentialcomponent of the virion) as illustrated in FIG. 12. Cotransfection ofsaid 293Cre cells with a plasmid containing a single lox P site upstreamof fibre, such as pFG23dX1lox, optionally carrying a fibre or E4 genemutation or insertion of foreign DNA, results in high-efficiencysite-specific recombination between the plasmid and viral DNA andresults in a virus whose fibre gene is derived from the plasmid asillustrated in FIG. 12 or FIG. 13. Therefore, it will be readilyappreciated by one skilled in the art that mutations, deletions or othermodifications engineered in and around the fibre gene of the plasmid,are rescued into the infectious virus genome. As an example, not meantto be limiting, the combination of plasmid, virus DNA and recombinase asillustrated in FIGS. 12 and 13 leads to high-efficiency substitution ofwild-type fibre with modified fibre genes for production of mutantviruses whose virion capsids contain altered fibre.

As a further example of the utility of this approach, a foreign DNAsegment is introduced into a plasmid, such as pFG23dX1lox, between thelox site and the coding sequences of fibre, such that said foreign DNAsegment is rescued into virus by cotransfection of 293Cre cells with DNAprepared from Adlox2fibre (FIG. 13). As in the examples describedpreviously for use of the two plasmid system, said foreign DNA segmentcould be a separate expression cassette or could be a fusion of peptidesequences such as a ligand to a cell surface receptor.

Table 5 provides results documenting the efficiency with which Cremediated recombination can be used to generate infectious virus bycotransfection of 293Cre cells as illustrated in FIG. 9. It is apparentthat the efficiency of rescue is comparable to that shown in Tables 1and 2 and is several fold higher than the efficiency of homologousrecombination (pFG173+pFG23dX1).

EXAMPLE 10 Use of Alternate Adenoviral Vector Systems According to thisInvention

Those skilled in the art will recognize, based on the instantdisclosure, that in the system described herein according to FIG. 8C,the left most lox site is not essential when the viral DNA is digestedwith enzymes such as those depicted, namely AsuII and/or SwaI. It willalso be recognized that enhanced rescue of mutations or inserts into theviral genome by cotransfection of cells with a plasmid plus a viral DNAfragment with TP does not require a TP at both ends so the large viralDNA fragment generated by AsuII and/or SwaI digestion and having a TP atthe right end only is sufficient for this system to operate efficiently.Similarly in the systems disclosed according to FIGS. 12 and 13, onlythe lox site 5′ of fibre is necessary if the viral DNA-TP is cleavedwith one or more enzymes that cut to the right, e.g. in fibre or in E4.If there are not naturally occurring restriction sites suitable for thispurpose, such sites may easily be engineered by those of ordinary skillin the art, based on the present disclosure. For example we haveidentified a Blp I site between the 3′ end of fibre and the codingsequences for E4 that can be used to insert a synthetic DNA. Asillustrated in FIG. 14 we inserted a lox DNA sequence into this site butwe could easily have introduced DNA containing a restrictionendonuclease site that is not present elsewhere in the viral genome, andsaid restriction site could be rescued into an infectious virus asillustrated in FIG. 15.

It will further be recognized, based on the present disclosure, that thecombination of Cre-lox with the two plasmid system will have widestapplication because of its simplicity: only readily prepared plasmid DNAis required, no restriction enzyme digestions are required, no possiblebackground of parental viruses has to be contended with, and the systemis more than adequately efficient for most purposes. Nonetheless, whenenhanced levels of infectivity are required, utilization of the methodsdisclosed herein for use of viral DNA incorporating bound terminalprotein may also benefit through combination with the site-specificrecombination techniques taught herein. TABLE 1 Cotransfections on 293and 293Cre cells for rescue of LacZ (±loxP) Plasmid μg Plaques/dishPlaques/dish combo DNA (293 cells) (Totals) (293Cre cells) (Totals)pCA36: 5:5 0, 0, 0, 0 (5) 0, 1, 2, 0 (7) pBHG10  5:10 0, 0, 0, 1 1, 0,0, 0 10:10 2, 0, 1, 1 1, 2, 0, 0 pCA36: 5:5 0, 0, 0, 1 (5) 0, 0, 0, 0(0) pBHG10lox  5:10 0, 0, 0, 1 0, 0, 0, 0 10:10 0, 0, 2, 1 0, 0, 0, 0pCA36lox: 5:5 1, 3, 1, 0 (6) 0, 1, 0, 1 (7) pBHG10  5:10 0, 1, 0, 0 0,0, 1, 2 10:10 0, 0, 0, 0 0, 1, 1, 0 pCA36lox: 5:5 1, 0, 0, 1 (4) 15, 14,20, 20 (168)  pBHG10lox  5:10 0, 0, 0, 0 11, 15, 12, 16 10:10 0, 0, 1, 118, 9, 10, 8

TABLE 2 Cotransfections on 293 and 293Cre cells for rescue of LacZ(±loxP) Plasmid μg Plaques/dish Plaques/dish combo DNA (293 cells)(Totals) (293Cre cells) (Totals) pCA36: 5:5 1, 1, 2, 6, (15) 1, 1, 2, 1,2,  (10) pBHG10lox 2, 3 3 pCA36lox: 5:5 1, 2, 2, 2, (10) 41, 44, 41,(242) pBHG10lox 2, 1 41, 44, 31 pCA36loxΔ: 5:5 0, 0, 0, 0,  (0) 41, 36,55, (230) pBHG10lox 0, 0 34, 24, 40 FG140 1 72, 72 150, 115

TABLE 3 Efficiency of Ad vector rescue by cotransfection with pBHG10loxand various shuttle plasmids^(a) Cell line Shuttle plasmid Plaques/dishAverage/dish 293 pCA36lox 6, 2, 3, 3, 5 3.8 pCA36loxΔ 1, 4, 0, 0, 0 1.0pCA36loxΔCreR 2, 2, 4, 3, 2 2.6 pCA36loxΔCreT 9, 4, 4, 7, 3 5.4 293CrepCA36loxΔ 23, 28, 22, 28 25.3^(a)5 μg of all plasmids were used in cotransfections.

TABLE 4 Efficiency of Ad vector rescue by cotransfection of 293 cellswith pBHG10lox and shuttle plasmids encoding Cre^(a). Cell line Shuttleplasmid Plaques/dish Average/dish 293 pCA36lox 2, 3, 1, 0, 1 1.4pCA36loxΔ 1, 0, 0, 0, 0 0.2 pCA36loxΔCreT^(b) 3, 1, 5, 2, 4 3.0pCA36loxCreITR^(b) 21, 20, 42, 34, 40 31.4^(a)All cotransfections performed with 5 μg of the indicated shuttleplasmid and 5 μg of pBHG10lox^(b)Plasmids illustrated in FIG. 8c.

TABLE 5 Efficiency of rescue of fibre and E4 genes into Ad bycotransfection with pFG173lox and pFG23lox^(a) μg Number of plaques(average/dish) Plasmids DNA 293 cells 293Cre cellspFG173lox^(b):pFG23dX1loxc^(c) 5:5 0, 0, 0, 0 (0) 33, 27, 39, 26 (31)2:2 0, 0, 0, 0 (0) 9, 15, 10, 9 (11) pFG173:pFG23dX1 5:5 0, 0, 0, 0 (0)0, 0, 1 (0.3) pFG140 1 95 93^(a)Cotransfections as diagrammed in FIG. 9^(b)Diagrammed in FIG. 9b^(c)Diagrammed in FIG. 10

1. A method for introducing and expressing a foreign nucleic acidsequence in a host cell, comprising: (a) introducing into a mammalianhost cell a first plasmid comprising a modified adenovirus genome havinga modification within early region 1 (E1) sufficient to render saidfirst plasmid unable to form viable viral particles in mammalian hostcells, said modification eliminating susceptibility of said firstplasmid to being encapsidated into a viral particle, said first plasmidfurther comprising at least one nucleic acid sequence for (A) encodingantibiotic resistance and (B) for replication of said modifiedadenovirus genome in bacterial host cells, and said first nucleic acidsequence further comprising at least one site-specific recombinaserecognition target site which is recognized by a site-specificrecombinase; (b) introducing into said host cell recited in (a), an E1shuttle plasmid that comprises a sequence comprising an adenovirusgenome E1 region, comprising a packaging signal, and further comprisingat least one recombinase recognition target site sufficiently identicalwith said recombinase recognition target site in said first nucleic acidas to be recognized by the same site-specific recombinase whichrecognizes said site-specific recombinase recognition target site insaid first nucleic acid, and an inserted foreign nucleic acid sequencecomprising a coding sequence having an open reading frame inserted intothe E1 region and comprising sequences that regulate the expression ofsaid open reading frame; and wherein a site-specific recombinase whichrecognizes said site-specific recombinase recognition target sites iseither (i) expressed by a cell into which said first and said secondnucleic acids are introduced, (ii) operatively encoded by said firstnucleic acid, said second nucleic acid or both, or (iii) is provided intrans through expression from a third nucleic acid or is provided intrans as an active protein; (c) isolating virus particles comprising arecombined modified adenoviral genome having elements of said firstplasmid plus the packaging signal and the inserted foreign nucleic acidsequence of the E1 shuttle plasmid; (d) introducing said recombinantmodified viral genome into a host cell; and (e) expressing the codingsequences contained in said modified recombinant viral genome.
 2. Themethod according to claim 1 wherein said at least one site-specificrecombinase recognition target site of said first nucleic acid sequenceand said at least one site-specific recombinase recognition target siteof said second nucleic acid sequence each is comprised of a loxPsequence, and wherein said site-specific recombinase is comprised ofCre.
 3. The method according to claim 1 wherein said at least onesite-specific recombinase recognition target site of said first nucleicacid sequence and said at least one site-specific recombinaserecognition target site of said second nucleic acid sequence each iscomprised of an frt sequence, and wherein said site-specific recombinaseis comprised of FLP.
 4. A method for eliciting an immune response in amammalian host to protect against an infection comprising administeringa vaccine composition comprising the recombined modified adenoviralgenome of claim
 1. 5. A vaccine for protecting a mammalian host againstinfection comprising: (a) the isolated virus particle of claim 1comprising the recombined modified adenoviral genome wherein theinserted foreign nucleic acid encodes an antigenic determinant producedby a disease organism responsible for the infection; and (b) apharmaceutically acceptable excipient.
 6. A composition comprising therecombination product of a first plasmid comprising a modifiedadenovirus genome having a modification within early region 1 (E1)sufficient to render said first plasmid unable to form viable viralparticles in mammalian host cells, said modification eliminatingsusceptibility of said first plasmid to being encapsidated into a viralparticle, said first plasmid further comprising at least one nucleicacid sequence for (A) encoding antibiotic resistance and (B) forreplication of said modified adenovirus genome in bacterial host cells,and said first nucleic acid sequence further comprising at least onesite-specific recombinase recognition target site which is recognized bya site-specific recombinase and a second plasmid that comprises asequence comprising an adenovirus genome E1 region, comprising apackaging signal, and further comprising at least one recombinaserecognition target site sufficiently identical with said recombinaserecognition target site in said first nucleic acid as to be recognizedby the same site-specific recombinase which recognizes saidsite-specific recombinase recognition target site in said first nucleicacid, and an inserted foreign nucleic acid sequence comprising a codingsequence having an open reading frame inserted into the E1 region andcomprising sequences that regulate the expression of said open readingframe; and wherein a site-specific recombinase which recognizes saidsite-specific recombinase recognition target sites is either (i)expressed by a cell into which said first and said second nucleic acidsare introduced, (ii) operatively encoded by said first nucleic acid,said second nucleic acid or both, or (iii) is provided in trans throughexpression from a third nucleic acid or is provided in trans as anactive protein.